Polypeptides are useful for the treatment of disease in humans and animals. Examples of such polypeptides include insulin for the treatment of diabetes, interferon for treating viral infections, interleukins for modulating the immune system, erythropoietin for stimulating red blood cell formation, and growth factors that act to mediate both prenatal and postnatal growth.
Many bioactive polypeptides can be produced through use of chemical synthesis methods. However, such production methods are often times inefficient and labor intensive which leads to increased cost and lessened availability of therapeutically useful polypeptides. An alternative to chemical synthesis is provided by recombinant technology which allows the high yield production of bioactive polypeptides in microbes. Such production permits a greater number of people to be treated at a lowered cost.
One obstacle to the administration of polypeptides to humans and animals is degradation. Many endogenous proteases exist in humans and animals that rapidly degrade foreign as well as native polypeptides. Degradation by these proteases reduces the effectiveness of therapeutically active polypeptides. One method that may be used to counter the effects of such proteases is to administer polypeptides having increased resistance to proteolytic degradation. Polypeptides having an increased resistance to proteolytic degradation may be produced by replacing a carboxyl group located on the carboxyl-terminus of the polypeptide with an amine group through a process of amidation.
Chemical amidation reactions have been used in the past to change the carboxyl-group to an amide group. Such methods involve the use of expensive and toxic chemicals as well as add a process step during the production of a therapeutically useful polypeptide. This added step reduces the yield of these therapeutically active polypeptides and increases their cost.
An alternative to the use of chemical amidation reactions is use of the protease clostripain. Clostripain (EC 3.4.22.8) is an endopeptidase that cleaves a polypeptide at the carboxyl-terminus of Arg residue. Accordingly, use of clostripain during the production of an amidated therapeutic polypeptide from a precursor polypeptide allows the precurser polypeptide to be cleaved and amidated in the same step to produce a therapeutic polypeptide.
Clostripain is expressed by the anaerobic bacteria Clostridium histoliticum and can be isolated from culture filtrates by conventional methods. However, isolation of clostripain from culture filtrates is expensive, inefficient, and is susceptible to contamination by other unwanted proteases that may adversely affect later use of clostripain during production of therapeutic polypeptides. Clostripain has also been expressed and isolated from Escherichia coli and Bacillus subtilis. However, these attempts produced low yields of clostripain that was of low enzymatic activity.
The ability to efficiently produce active clostripain on a large scale would allow for the more efficient production of therapeutic polypeptides at a lessened cost. Accordingly, a need exists for efficient production methods to produce clostripain.